thymoquinone and bronchodilation: the possible mechanism and … · issues biol. sci. pharma. res....

Issues in Biological Sciences and Pharmac eutical Research Vol.8 (1),pp.1-19, February 2020 Available online at https ://www.journalissues.org/IBSPR/ https://doi.org/10.15739/ibspr.20.001 Copyright © 2020 Author(s) retain the copyright of this article ISSN 2350-1588

Original Research Article

Thymoquinone and Bronchodilation: The possible mechanism and therapeutic potential of an emerging

natural drug in reactive airway disease

Received 15 November, 2020, Revised 19 December, 2019 Accepted 28 December, 2019 Published 7 January, 2020

Adnan Bashir1, Yasir Arfat2*,

Madiha Rasheed3, Salman Iftikhar4,

Rao Salman Aziz4, Maheen Rana5 and

Muhammad Rashid6

1Department of Pharmacology, Fatima Memorial College of Medicine and

Dentistry Lahore, Pakistan 2King College of Bioresources Chemical

and Material Engineering, Shaanxi University of Science and Technology,

Xian, China. 3Department of Oral Biology, Wateem Dental College Rawalpindi, Pakistan

4Department of Pharmacology, Rashid Latif Medical and Dental College Lahore,

Pakistan. 5Department of Pathology, Rashid Latif

Medical and Dental College Lahore, Pakistan.

6Faculty of Fisheries and Wildlife, University of Veterinary and Animal

Sciences, Lahore, Pakistan.

*Corresponding Author Email: [email protected]

Tel.:+86-13022970072

INTRODUCTION

Thymoquinone has a quantitative relaxant effect on the tracheal sm ooth muscle. In this study, we also attempted to investigate the probable mechanism of action of thymoquinone by predisposing the tracheal tissue of our experimental model to carbachol, a muscarinic agonist that produces contractions in the tissue and propranolol which blocks receptors. The study was carried out on tracheal tissue from two groups of guinea pigs. Group 1 served as normal, while group 2 was sensitized and stimulated with ovalbumin to create airway hyper-responsiveness. We compared the percentage of relaxation produced by thymoquinone and salbutamol. It was observed that in the presence of high concentrations of the agonist, thymoquinone produced more relaxation in both sets of the experiment. It caused relaxation in the presence of propranolol, but it was not completely reversed. The relaxation produced by thymoquinone was also compared to salbutamol (conventiona lly used Beta agonist in reactive airway disease) and was observed to be less. Our results suggest that thymoquinone has properties of the muscarinic blockade and, among other mechanisms involved in the relaxation of smooth muscles relaxation, also has 2 agonistic activity. Key words: Thymoquinone, ovalbumin, salbutamol, carbachol, propranolol, reactive airway disease.

ABBREVIATIONS PGMI: Postgraduate Medical Insti tute Lahore, Pakistan UHS: University of Health Sciences Lahore, Pakistan PBS: Phosphate Buffer Saline Al(OH)3: Aluminum Hydroxide: SEM: Standard Error of the Mean SPSS: Statistical Package for the Social Sciences

Bronchial asthma is a chronic inflammatory disease of the respiratory tract,which is characterized by episodic symptoms such as shortness of breath, coughing and wheezing and can increase and decrease over time.

Approximately 8.2% of the general population is affected. The prevalence increased from 2005 to 2009 and is now at the highest level (Akinbami et al., 2012). Asthma is responsible for considerable morbidity and health-care

Issues Biol. Sci. Pharma. Res. 2 costs. Significant progress was made in important outcomes such as hospital admissions with asthma and mortality in the 1990s and early 2000s, but littl e improvement has been observed in the past 10 years, despite rising treatment costs. There are no techniques for evaluating incipient, and incipient disclosure has progressed more slowly than in other specialties(Pavord et al., 2018). If asthma with an underlying inflammatory pathophysiology is left untreated, the airway can be remodeled, resulting in increased severity and incidence of exacerbations or deaths(Bush 2019). Asthma deaths are rel atively rare, but according to the American Academy of Asthm a Allergy and Immunology, they caused 3,447 deaths in 2007 i.e., bout 9 a day in the U.S.(Akinbami et al., 2011). Most of the asthmatic medicines work by relaxing bronchioles or by reducing the inflammation. The dugs include bronchodilators like beta 2 receptor agonist, anticholinergics, methylxanthines and anti -inflammatory agents like inhaled and systemic corticosteroids, mast cell stabilizers, leukotriene antagonists, lipoxygenase inhibitors and anti-IGE monoclonal antibodies (Sehmi et al., 2016).The al ternative therapies most commonly used to treat asthma are dietary changes, herbal remedies, meditation and homeopathy. Andhra Pradesh's tribal and non-tribal people used almost 80 medicinal plants to treat asthma. The tribal people firmly believe in the traditional health system through herbal treatment (Peprah et al., 2018; Savithramma et al., 2007). Nigella sativa seeds are usually used in the Arab world as a traditional medicine for the treatment of various diseases. Recently, the commercial use of these seeds has increased in Southeast Asia due to the presence of important trace elements such as zinc and magnesium, which are beneficial for improving immunity levels. They are therefore helpful in preventing asthma (Shomar, 2012; Yimer et al., 2019).Different extracts of Nigella Sativa have a bronchodilator effect due to different mechanisms of action. The aqueous and macerated extracts of Nigella Sativa show a bronchodilatory effect of this plant due to their calcium channel blocking properties (Boskabady et al., 2004b). The boiled extract of this plant shows a significant rel axation of the smooth breathing muscles in asthmatics (Boskabady et al., 2007).Nigella Sativa volatile oil has been reported to be used in many patients suffering from inflammatory respiratory disease because it reduces airway infl ammation and has a relaxing effect on the respiratory system (El Tahir et al., 1993; Mahboubi, 2018). Nigella sativaoil and boiled extract are suitabl e for the prophylaxis and treatment of an acute asthma attack (El Aziz et al., 2011; Ikhsan et al., 2018).Thymoquinone is the main component of this herb, and its effect on the prevention of respiratory infl ammation in the animal model of asthma is known and shows a decrease in lung eosinophilia and serum levels of antibodies,(El Gazzar et al., 2006a) and an inhibition of lipoxygenase expression by lung cells, which leads to a decrease in the level of leukotriene,(El Gazzar et al., 2006b)and a decrease in the response of the trachea to methacholine (Keyhanmanesh et al., 2010).

Their rol e in the treatment of respiratory distress

syndromes was rated as beneficial (Gholamnezhad et al., 2019; Isik et al., 2005). Studies show that the preventive and anti-inflammatory effects of thymoquinone are roughly equivalent to steroids(Keyhanmanesh et al., 2010).It reduces the release of mediators that are involved in chronic respiratory infections, such as histamine, serotonin and bradykinin (El Gazzar et al., 2006b), it has an anti tussive effect, which is likely to occurs through opioids receptors (Hosseinzadeh et al., 2008).The effect of thymoquinone on the contraction of the smooth muscles of the normal guinea pig trachea was examined and showed relaxation (Al-Majed et al., 2001).

Other observed effects and traditional uses of Nigella sativa include Anti-hypertensive and diuretic effect, (Leong et al., 2013) anti- inflammatory activity,(Bayir et al., 2012) antidepressant activity,(Perveen et al., 2009) anthelmintic activity,(Shalaby et al., 2012) anti-seizures activity,(Arafa et al., 2013) antibacterial and anti-fungal activity,(Haloci et al., 2012) anti-cancer activity,(Randhawa and Alghamdi 2011) antispasmodic and antiulcer effects(Rajkapoor et al., 2002).The aim of the present study was to examine the effect of thymoquinone on carbachol-induced tracheal smooth muscle contractions in normal and ovalbumin-sensitized guinea pig trachea and to find out possible mechanisms of action. MATERIALS AND METHODS Animals 25 animals kept in each group included a total of total 2 groups, one normal and another ovalbumin sensitized guinea pigs were asthmatics. we used thymoquinone as a research drug Krebs solution to keep the tracheal tissue vital to the functioning of solution containing carbachol to induce the contraction of tissue salbutamol as a medication to compare relaxation. All the analysis performed on computerized data monitoring device “POWERLab”. The study was conducted at the Postgraduate Medical Insti tute (PGMI), Lahore and University of Health Sciences (UHS), Lahore, Pakistan. Isolated pieces of guinea pig tracheal tissue were harvested and randomly sampled by using lottery method. Tissues that respond equally to 160 nM carbachol concentrations applied consecutively at 5 min intervals with intermediate washing were considered stable and selected for the experiment. Healthy guinea pigs weighing 250 to 300g were procured from the University of Veterinary and Animal sciences Lahore, Pakistan. Animals were kept in the PGMI animal house, at a temperature of 22-24°C. They were kept under natural light and dark cycles and fed with vegetables, fruits and water ad libitum. In experimental group 2, allergic airway inflammation was induced by sensitization and airway irritation, whereas normal healthy (group 1) adult guinea pigs were not exposed to any chemical or allergen. For sensitization, 100µg of ovalbumin was given with 200µg of Al(OH)3 in phosphate buffer saline (PBS), administered by

Bashir et al. 3

Table 1. Effect of carbachol on normal guinea pig tracheal tissue (n=6)

Conc. Carbachol in Nanomolar 80 320 1300 Log Concentration 1.9 2.5 3.1 Response of Experiments (Force of contraction in mN) 1 21.6 35.50 57.50 2 19.9 31.6 51.3 3 6.3 15.5 38.9 4 8.2 27.6 55.0 5 7.7 18.7 44.9 6 13.0 29.3 53.6 Mean response ±SEM (mN) 12.8±2.7 26.4± 3.2 50.2±2.9

intraperitoneal injections on day 0 and 14. The airways were challenged with 1% ovalbumin in PBS through nasal inhalation on day 25, 26 and 27.(Chang et al., 2011). Experiment Preparation Nasal inhalation airway challenge was achieved with 1% ovalbumin in P.B.S. solution through nasal inhalation. Preparation of ovalbumin solution: For 1% solution 1 g of ovalbumin was dissolved in a final volume of 100 ml of PBS. A nebulizer (M edicure, China) was used for nasal inhalation. The solution was put in the chamber of nebulizer. Each animal was nebulized for twenty minutes on day 27, 28, 29. Animals were sacrificed by over dosage of chloroform on day 28 i.e., 24 hr after last challenge. Tracheal tissues were dissected out and kept in Kreb’s solution. Each tissue was cleaned from the surrounding fatty tissues. The tracheal tube was cut into two 5-6 mm wide rings, each containing about five to six cartilages. Each ring was opened by a longitudinal cut on the ventral side opposite to smooth muscle layer, which formed a tracheal strip, with a central part of smooth the muscle enclosed between the cartilaginous parts at the edges (Keyhanmanesh et al., 2013). Experimental Procedure The isolated tissue preparation was mounted in a 50 ml tissue bath containing the Kreb’s solution with the composition (mM): NaCl 118.2, NaHCO3 25.0, CaCl2 2.5, KCl 4.7, KH2PO4 1.2 and glucose 11.7; maintained at 370C and aerated with oxygen gas. A tension of 1g was applied to each of tracheal strip and held constant during the experiment. The tissue was equilibrated for one hour before starting experiment with a solution change every 15 minutes. The tracheal contraction was recorded with an isometric transducer on a computerized data recording system (PowerLab) and the responses measured in millinewtons (mN). Statistical Analysis The data were analyzed with SPSS (Statistical Package for the Social Sciences), version 20. They are given as the mean

± SEM. The percentage decrease in response is calculated using different concentrations of each drug. The t-test with two paired tail samples is used to compare the responses with different concentrations within the groups. A p-value less than or equal to 0.05 is considered significant. RESULTS

Effect of Carbachol on Guinea Pig Tracheal Tissue Graded concentrations of carbachol were applied cumulatively to the tissue untill the maximum effect was observed. Each concentration was left to act for one and a half minute. Following concentrations, selected by preliminary experiments, were used i.e., 10 nm, 20 nm, 40 nm, 80 nm, 160 nm, 320 nm, 640 nm, 1300 nm, 2600 nm and 5200 nm. The experiment was performed on tissues from six animals and the response to each concentration was shown in mN measured as indicated in supplementary Table 1. The data were expressed as mean ± SEM and recorded to plot a concentration response curve as shown in Figure 1.

Effect of Carbachol in the Presence of Thymoquinone on Guinea Pig Tracheal Tissue. Graded concentrations of carbachol, as used in experiment 1, were cumulatively applied to the tissue in the absence of thymoquinone. Then the same concentrations were applied to the same tissue in the presence of three increasing concentrations of thymoquinone selected by preliminary experiments (122 µM, 244 µM and 488 µM) and the effect was recorded. Tissue was washed three times and given a 30-minute break between each set of responses. During that period Krebs’s solution was changed every 10 min. Th e experiment was performed on tissues from six animals and response to each concentration was measured in mN, as given in supplementary Table 2. The data was expressed as mean ± SEM and recorded to plot concentration response curves to see the shift as shown in Figure 2. Three concentrations which gave 25%, 50 % and 75 % of the maximum response, were selected for statistical analysis.

Issues Biol. Sci. Pharma. Res. 4

Figure 1: Concentration response curve of carbachol to normal guinea pig tracheal tissue. The data represents mean ±SEM (n=6). The maximum response with carbachol in sensitized tissue was assumed to be 100%.

Effect of Carbachol in the Presence of Thymoquinone and Propranolol on Guinea Pig Tracheal Tissue Concentrations of carbachol as used in Experiment 1 were cumulatively applied to the tissue. The tissue was preincubated with propranolol (68 nM) and the same concentrations of carbachol were applied to same tissue in the presence of three increasing concentrations of thymoqui none selected by preliminary experiments (122 µM, 244 µM and 488 µM) effect was recorded. The tissue was washed three times and there was a 30 minute pause between each set of responses. During this time Kreb’s solution was changed every 10 minutes. The experiment was carried out on tissues from six animals and response to each concentration was measured in mN as shown in supplementary Table 3. The data was expressed as mean ± SEM and recorded to plot concentration response curves to see the shift as shown in Figure 3. Three concentrations which gave 25%, 50 % and 75 % of the maximum response, were selected for statistical analysis. Effect of Carbachol in the Presence of Salbutamol on Guinea Pig Tracheal Tissue Concentrations of carbachol as used in the above experiments were cumulatively applied to the tissue. Then the same concentrations of carbachol were applied to same tissue in the presence three increasing Salbutamol concentrations selected by preliminary experiments (17 nM, 34 nM and 68 nM) and the effect was recorded. The tissue was washed three times and there was a 30-minute pause between each set of responses. During this time, Kreb's solution was changed every 10 minutes. The experiment was carried out on tissues from six animals and the respons e to each concentration was measured in mN as shown in supplementary Table 4.The data was expressed as mean ± SEM and recorded to plot concentration response

curves to see the shift as shown in Figure 4. Three concentrations, which gave 25%, 50% and 75% of the maximum response, were selected for statistical analysis. Effect of Carbachol in the Presence of Salbutamol and Propranolol on theTracheal Tissue of Guinea Pig Concentrations of carbachol as used in above experiments were cumulatively applied to the tissue. Tissue was pre-incubated with propranolol selected by preliminary experiments (68 nM) and equal concentrations of carbachol were applied to same tissue in the presence of three increasing concentrations of salbutamol and the effect was recorded. The tissue was washed three times and there was a 30-minute pause between each set of responses. During this time, Kreb's solution was changed every 10 minutes. The experiment was carried out on tissues from six animals and the response to each concentration was measured in mN as given in supplementary Table 5. The data was expressed as mean ± SEM and used to plot the concentration response curves to see the shift as shown in Figure 5. Three concentrations which gave 25%, 50 % and 75 % of maximum response, were selected for statistical analysis.

Effect of Thymoquinone on Carbachol-Induced Contraction of Ovalbumin-Sensitized Guinea Pigs Tracheal Tissue

Graded concentrations of carbachol as used in experiment 1 were cumul atively applied to theovalbumin-sensitized tissue in the absence of thymoquinone. Then same concentrations were applied to the same tissue in the presence of three increasing concentrations of thymoquinone selected by preliminary experiments (122 µM, 244 µM and 488 µM) and the effect was recorded. The

Bashir et al. 5

Table 2. Effect of thymoquinone (µM) on carbachol (nM) induced contraction of guinea pig tracheal tissue

Carbachol (nM)

Carbachol (nM) and

Thymoquinone 122 (µM)

Carbachol (nM) and

Thymoquinone 244 (µM)

Carbachol (nM) and

Thymoquinone 488 (µM) Conc. Carbachol in Nanomolar 80 320 1300 80 320 1300 80 320 1300 80 320 1300 Log Concentration 1.9 2.5 3.1 1.9 2.5 3.1 1.9 2.5 3.1 1.9 2.5 3.1 Response of Experiments (Force of contraction in mN)

1 21.6 35.5 57.5 8.8 20.9 50.4 7.1 19.7 45.1 7.5 11.9 29.6 2 19.9 31.6 51.3 11.5 38.3 46.5 5.3 16.4 32.6 6.7 17.6 26.1 3 6.3 15.5 38.9 9.7 22.9 35.5 4.7 14.5 36.0 4.1 13.3 22.3 4 8.2 27.6 55.0 11.6 25.7 44.0 8.7 19.0 29.9 2.3 12.1 17.2 5 7.7 18.7 44.9 6.5 28.0 47.3 7.3 23.8 37.4 2.0 15.7 25.9 6 13.0 29.3 53.6 8.5 23.2 59.2 6.1 10.7 28.9 4.7 11.5 31.5 Mean response ± SEM (mN) 12.8±2.7 26.4 ±3.2 50.2±2.9 9.4±0.8 26.5±2.6 47.2±3.2 6.5±0.6 17.4±1.8 35.0±2.4 4.5±0.9 13.7±1.0 25.4±2.1 Mean decrease in response ± SEM (mN)

- - - 3.4±2.8 0.13±4.1 3.1±4.3 6.3±2.8 9.0±3.7 12.2±3.8 8.2±2.8 12.7±3.3 24.8±3.5

% age decrease in response - - - 26.6 0.5 6.2 49.2 34.1 24.3 64.1 48.1 49.4

P – value - - - 0.263 0.974 0.277 0.078 0.065 0.001*** 0.007** 0.014* 0.000***

Results of six experiments with mean± SEM, mean decrease in response, % decrease in response as well as level of significance as calculated by two tailed paired sample t-test

Figure 2: Concentration response curve of carbachol-induced contraction of guinea pig tracheal tissue in the presence of thymoquinone (µM). The data represents the mean ±SEM(n=6).The maximum response with carbachol in sensitized tissue was assumed to be 100%.

***


Table 3. Effect of thymoquinone (µM) and propranolol (nM) on carbachol (nM) induced contraction of guinea pig tracheal tissue

Carbachol (nM)

Carbachol (nM), Thymoquinone 122 (µM) and Propranolol 68 (nM)



Conc. Carbachol in Nanomolar 80 320 1300 80 320 1300 80 320 1300 80 320 1300 Log Concentration 1.9 2.5 3.1 1.9 2.5 3.1 1.9 2.5 3.1 1.9 2.5 3.1 Response of Experiments (Force of contractionin mN) 1

21.6 35.5 57.50 5.0 15.6 41.0 9.2 21.7 47.2 6.3 22.8 40.7

2 19.9 31.6 51.3 6.1 20.3 36.0 9.1 25.2 41.6 4.7 22.1 32.6 3 6.3 15.5 38.9 4.6 13.3 40.5 6.7 16.8 38.3 9.6 17.6 28.0

4 8.2 27.6 55.0 8.2 14.9 36.8 8.2 23.2 35.9 8.0 16.5 33.0 5 7.7 18.7 44.9 9.0 19.6 34.5 9.7 20.2 39.3 6.2 19.9 35.9 6 13.0 29.3 53.6 8.4 18.6 44.9 10.8 24.3 43.3 9.4 22.0 35.1 Mean response ± SEM (mN) 12.8±2.7 26.4±3.2 50.2±2.9 6.9±0.8 17.0±1.2 39.0±1.6 8.9±0.6 21.9±1.3 40.9±1.6 7.4±0.8 20.2±1.1 34.2±1.7 Mean decrease in response ± SEM (mN)

- - - 5.9±2.8 9.3±3.4 11.3±3.3 3.8±.2.8 4.5±3.4 9.3±3.3 5.4±2.8 6.2±3.3 16± 3.3

% age decrease in response - - - 47.1 35.2 22.5 29.7 17.0 18.5 42.2 23.5 31.9 P – value

0.113 0.029 0.013 0.189 0.111 0.014* 0.156 0.062 0.001***

Results of six experiments with mean ± SEM, mean decrease in response, % decrease in response as well as level of significance as calculated by two tailed paired sample t-test.

Figure 3: Concentration response curve of carbachol induced contraction on guinea pig tracheal tissue in the presence of thymoquinone (µM) and propranolol (nM). The data represents mean ±SEM (n=6). The maximum response with carbachol in sensitized tissue was assumed to be 100%.

Bashir et al. 7

Table 4. Effect of salbutamol (nM) on carbachol (nM) induced contraction of guinea pig tracheal tissue

Carbachol (nM)

Carbachol (nM) and Salbutamol 17 (nM)



Conc. Carbachol in Nanomolar 80 320 1300 80 320 1300 80 320 1300 80 320 1300

Log Concentration 1.9 2.5 3.1 1.9 2.5 3.1 1.9 2.5 3.1 1.9 2.5 3.1 Response of Experiments (Force of contraction in mN) -1

21.6 35.5 57.5 5.8 11.6 25.9 5.3 16.8 35.4 3.5 13.4 20.3

2 19.9 31.6 51.3 6.2 21.6 31.8 5.0 15.8 26.6 2.4 10.6 20.1 3 6.3 15.5 38.9 5.7 19.6 34.9 5.5 14.0 29.5 4.2 8.5 18.5 4 8.2 27.6 55.0 6.6 14.5 36.6 4.0 10.1 22.4 3.0 8.0 14.9 5 7.7 18.7 44.9 9.7 20.3 31.2 3.7 13.2 20.2 2.8 9.0 16.3 6 13.0 29.3 53.6 8.0 20.0 32.3 1.7 9.9 25.3 2.6 9.7 15.9 Mean response ± SEM (mN) 12.8±2.7 26.4±3.2 50.2±2.9 7.0±0.6 17.9±1.6 32.1±1.5 4.2±0.6 13.3±1.2 26.6±2.2 3.1±0.3 9.9±0.8 17.7±0.9 Mean decrease in response ± SEM (mN)

- - - 5.8±2.8 8.4±3.5 18.1±3.2 8.6±2.8 13.1±3.4 23.6±3.6 9.7±2.7 16.5±3.3 32.5±3.0

% age decrease in response - - - 45.3 31.8 36.1 67.2 49.6 47.0 75.8 62.5 64.7 P – value - - - 0.111 0.099 0.005** 0.022* 0.008** 0.001*** 0.018* 0.002** 0.000****

Figure 4: Concentration response curve of carbachol-induced contraction on guinea pig tracheal tissue in the presence of salbutamol (nM).The data represent the mean ±SEM (n=6).The maximum response with carbachol in sensitized tissue was assumed to be 100 %.


Table 5. Effect of salbutamol (nM) and propranolol (nM) on carbachol (nM) induced contraction of guinea pig tracheal tissue

Carbachol (nM)

Carbachol (nM), Salbutamol 17 (nM) and Propranolol 68 (nM)



Conc. Carbachol in Nanomolar 80 320 1300 80 320 1300 80 320 1300 80 320 1300

Log Concentration 1.9 2.5 3.1 1.9 2.5 3.1 1.9 2.5 3.1 1.9 2.5 3.1 Response of Experiments (Force of contraction in mN)1

21.6 35.5 57.5 9.0 19.2 25.7 7.3 22.6 35.3 5.7 12.3 29.9

2 19.9 31.6 51.3 7.0 13.3 22.2 7.2 23.2 37.0 3.3 14.7 28.6

3 6.3 15.5 38.9 7.6 13.6 20.3 5.3 20.5 35.2 5.8 16.1 27.9 4 8.2 27.6 55.0 7.3 14.7 19.4 5.5 20.4 37.2 7.7 18.0 31.6 5 7.7 18.7 44.9 7.4 11.9 25.9 8.5 17.5 30.4 5.5 20.1 35.4 6 13.0 29.3 53.6 8.5 12.1 24.8 7.3 22.6 35.3 6.1 11.8 30.9 Mean response ± SEM (mN) 12.8±2.7 26.4±3.2 50.2±2.9 7.8±0.3 14.1±1.1 23.1±1.2 6.8±0.5 21.1±0.9 35.1±1.0 5.7±0.6 15.5±1.3 30.7±1.1 Mean decrease in response ± SEM (mN)

- - - 5.0±2.7 12.2±3.3 27.2±3.1 5.9±2.7 5.2±3.3 15.1±3.0 7.1±12.8 10.9±3.4 19.5±3.1

% age decrease in response - - - 39.1 46.2 54.2 46.1 19.7 30.1 55.5 41.3 38.8

P – value - - - 0.111 0.006** 0.000*** 0.068 0.096 0.002** 0.067 0.047* 0.001***

Results of six experiments with Mean ± SEM, mean decrease in response, % decrease in response as well as level of significance as calculated by two tailed paired sample t-test

Figure 5: Concentration response curve of carbachol-induced contraction on guinea pig tracheal tissue in the

presence of salbutamol (nM) and propranolol (nM). The data represent mean ±SEM (n=6). The maximum response with carbachol in sensitized tissue was assumed to be 100 %.

Bashir et al. 9

Table 6. Effect of thymoquinone (µM) on carbachol (nM) induced contraction of ovalbumin sensitized guinea pig tracheal tissue

Carbachol (nM)

Carbachol (nM) and Thymoquinone 122 (µM)



Conc. Carbachol in Nanomolar

80 320 1300 80 320 1300 80 320 1300 80 320 1300

Log Concentration 1.9 2.5 3.1 - - - - - - - - - Response of Experiments (Force of contraction inmN)-1

35.8 58.5 84.2 23.2 35.9 67.6 23 44.2 58.5 20.7 29.3 46.7

2 24 40.4 67.4 19.6 32 51.4 18.8 34.7 54.1 21.6 32.1 39.7 3 29.8 44.4 62.0 23.9 43.6 63 20.5 34.9 46.0 21.1 35.5 47.6 4 33.1 55.6 71.4 25.4 40.4 61.6 25.2 35.0 48.6 20.8 36.8 50.5 5 36.7 53.4 65.3 18.5 40.9 60.4 21.4 35.7 42.6 16.0 32.4 49.9 6 23.2 30.8 62.7 29.4 51.3 65.6 18.4 40.4 52.3 22.9 37.5 49.6 Mean response ± SEM (mN)

30.4±2.4 47.2±4.3 68.8±3.4 23.3±1.6 40.7±2.7 61.6±2.3 21.2±1.1 37.5±1.6 50.4±2.4 20.5±1.0 34.0±1.3 47.3±1.6

Mean decrease in response ± SEM (mN)

- - - 7.1±3.4 6.5±6.2 7.2±3.4 9.2±1.7 9.7±4.4 18.5±2.5 9.9±3.1 13.3±5.1 21.5±3.9

% age decrease in response

- - - 23.4 13.8 10.5 30.3 20.6 26.9 32.6 28.2 31.3

P – value - - - 0.089 0.339 0.089 0.003** 0.081 0.001*** 0.026* 0.049* 0.003**


tissue was washed three times and given a 30-minute pause between each set of responses. During that period Krebs’s solution was changed every 10 min. The experiment was carried out on tissues from six animals and the response to each concentration was measured in mN as given in supplementary Table 6. The data was expressed as mean ± SEM and recorded to plot concentration response curves to see the shift as shown in Figure 6. Three concentrations, which gave 25%, 50 % and 75 % of the maximum response were selected for statistical analysis. Effect of Thymoquinone and Propranolol on Carbachol-Induced Contractions in Ovalbumin-Sensitized Guinea Pig Tracheal Tissue Concentrations of carbachol as used in experiment 1

were cumul atively applied to the ovalbumin- sensitized tissue. Tissue was pre incubated with propranolol (68 nM) and the same concentrations of carbachol were applied to same tissue in the presence of three increasing concentrations of thymoquinone selected by preliminary experiments (122 µM, 244 µM and 488 µM) and the effect was recorded. The tissue was washed three times and there was a 30-minute pause between each set of responses. During this time, Kreb's solution was changed every 10 minutes. The experiment was carried out on tissues from six animals and the response to each concentration was measured in mN as given in supplementary Table 7. The data was expressed as mean ± SEM and recorded to plot concentration response curves to see the shift as shown in Figure 7. Three concentrations producing,

which gave 25%, 50 % and 75 % of maximum response, were selected for statistical analysis. Effect of Salbutamol on Carbachol-Induced Contractions of Ovalbumin -Sensitized Guinea Pig Tracheal Tissue. Concentrations of carbachol as used in above experiments were cumulatively applied to the ovalbumin-sensitized tissue. Then the same carbachol concentrations were applied to the same tissue in the presence of three increasing salbutamol concentrations selected by preliminary experiments (17 nM, 34 nM and 68 nM) and the effect was recorded. Tissue was washed three times and there was a 30-minute pause between each set of responses. During this time, Kreb's solution was


0.0

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Log concentration of carbachol (nM)

Ovalbuman Carbachol (nM) Carbachol (nM) + Thymoquinone 122 µM

Carbachol (nM) + Thymoquinone 244 µM Carbachol (nM) + Thymoquinone 488 µM

*****

***

Figure 6 : Concentration response curve of carbachol-induced contraction on ovalbumin-sensitized guinea pig tracheal tissue in the presence of thymoquinone (µM). The data represent the mean ±SEM ( n=6).The maximum response with carbachol in sensitized tissue was assumed to be 100%.

changed every 10 minutes. The experiment was carried out on tissues from six animals and the response to each concentration was measured in mN as given in supplementary Table 8. The data was expressed as mean ± SEM and recorded to plot concentration response curves to see the shift as shown in Figure 8. Three concentrations, which gave 25%, 50 % and 75 % of maximum response, were selected for statistical analysis. Effect of Salbutamol and Propranolol on Carbachol-Induced Contractions of Ovalbumin-Sensitized Guinea Pig Tracheal Tissue Concentrations of carbachol as used in the above experiments were cumulatively applied to the ovalbumin-sensitized tissue. The tissue was pre incubated with propranolol selected by preliminary experiments (68 nM) and equal concentrations of carbachol were applied to the same tissue in the presence of three increasing concentrations of salbutamol and the effect was recorded. Tissue was washed three times and there was a 30-minute pause between each set of responses. During this time, Kreb's solution was changed every 10 minutes. The experiment was carried out on tissues from six animals and the response to each concentration was measured in mN as given in supplementary Table 9. The data was expressed as mean ± SEM and was used to plot concentration response curves to see the shift as shown in Figure 9. Three concentrations,which gave 25%, 50 % and 75 % of the maximum response, were selected for statistical analysis. Comparison of Relaxant Effect of Thymoquinone on Normal Vs Ovalbumin Sensitized Guinea Pig Tracheal Tissue In normal tissue, the relaxing effects of thymoquinone at

122 µM on carbachol (80, 320 and 1300 nM) resulted in a 26.6, 0.5 and 6.2% decrease in contraction concentrations, respectively. While in ovalbumin-sensitized tissue, the same concentration of thymoquinone reduced responses to 23.4, 13.8 and 10.5%. The second concentration of thymoquinone 244 µM on carbachol (80, 320 and 1300 nM) resulted in a decrease in the contraction concentrations in normal tissue by 49.2, 34.1 and 24.3%, respectively. While in ovalbumin-sensitized tissue, the same concentration of thymoquinone reduced responses to 23.4, 13.8 and 10.5%. Similarly, the third concentration of thymoquinone 488 µM on carbachol (80, 320 and 1300 nM) resulted in a 49.2, 34.1 and 24.3% decrease in normal tissue contraction concentrations, respectively. While in ovalbumin-sensitized tissue, the same concentration of thymoquinone reduced responses to 32.6, 28.2 and 31.3%, as shown in Figure 10 and supplementary Table 10. Comparison of Relaxant Effect of Thymoquinone and Salbutamolon Ovalbumin -Sensitized Guinea Pig Tracheal Tissue In normal tissue, 17 nM concentration of Salbutamoland thymoquinone (concentration of 244 µM) as well as carbachol (80, 320 and 1300 nM) led to a decrease in the contraction concentrations by 23.4, 13.8 and 10.5%. While the same concentration of salbutamol thymoquinone and carbachol applied to ovalbumin-sensitized tissue could not prevent a decreasing concentration effect (41.4, 28.0 and 25.9%, respectively). In normal tissue, 34 nM concentrations of salbutamol and thymoquinone (concentration of 244 uM) and carbachol (80, 320 and 1300 nM) led to a decrease in the contraction concentrations by 30.3, 20.6 and 26.9%. While the same concentration of salbutamol thymoquinone and carbachol applied to ovalbumin-sensitized tissue could not prevent a decreasing

Bashir et al. 11 Table 7. Effect of thymoquinone (µM) and propranolol (nM) on carbachol (nM) induced contraction of ovalbumin sensitized guinea pig tracheal tissue

Carbachol (nM) Carbachol (nM), Thymoquinone

122 (µM) and Propranolol 68 (nM)

Carbachol (nM), Thymoquinone 244 (µM) and

Propranolol 68 (nM)

Carbachol (nM), Thymoquinone 488 (µM) and Propranolol 68

(nM) Conc. Carbachol in Nanomolar 80 320 1300 80 320 1300 80 320 1300 80 320 1300 Log Concentration 1.9 2.5 3.1 - - - - - - - - - Response of Experiments (Force of contraction in mN) 1

35.8 58.5 84.2 29.3 47 59.7 20.3 43.3 56.7 22.7 40.7 57.8

2 24 40.4 67.4 26.4 49.7 63.3 24.8 43.4 59.2 19.6 38.7 54.9

3 29.8 44.4 62.0 27.0 32.5 57.3 32.0 35.8 55.5 20.1 37.6 53.7

4 33.1 55.6 71.4 27.0 47.3 64.5 23.1 39.0 62.5 22.9 38.5 53.5

5 36.7 53.4 65.3 22.7 30.7 52.3 25.0 38.1 59.8 23.5 40.6 56.9

6 23.2 30.8 62.7 25.8 37.6 51.1 28.3 41.5 52.1 21.7 36.5 50 Mean response ± SEM (mN) 30.4±2.4 47.2±4.3 68.8±3.4 26.4±0.9 40.8±3.4 58.0±2.3 25.6±1.7 40.2±1.2 57.6±1.5 21.8±0.7 38.8±0.7 54.5±1.1 Mean decrease in response ± SEM (mN)

- - - 4.1±2.6 6.4±5.0 10.8±3.1 4.9±3.5 7.0±4.6 11.2±3.3 8.7±1.9 8.4±3.8 14.4±2.8

% age decrease in response - - - 13.5 13.6 15.7 16.1 14.8 16.3 28.6 17.8 20.9 P – value - - - 0.173 0.257 0.018 0.224 0.191 0.02* 0.077 0.007** 0.004**

Table 8. Effect of salbutamol (nM) on carbachol (nM) induced contraction of ovalbumin sensitized guinea pig tracheal tissue

Carbachol (nM) Carbachol (nM) and Salbutamol

17 (nM) Carbachol (nM) and Salbutamol

34 (nM) Carbachol (nM) and Salbutamol

68 (nM) Conc. Carbachol in Nanomolar 80 320 1300 80 320 1300 80 320 1300 80 320 1300 Log Concentration 1.9 2.5 3.1 - - - - - - - - - Response of Experiments (Force of contraction in mN) -1

35.8 58.5 84.2 18.5 37.0 51.8 20.0 28.8 39.9 12.5 22.4 33.7

2 24.0 40.4 67.4 17.8 31.8 52.8 16.0 27.0 40.0 13.5 24.5 32.0 3 29.8 44.4 62.0 16.4 35.0 49.3 16.5 28.4 40.6 15.6 26.3 34.7

4 33.1 55.6 71.4 18.5 33.2 50.7 14.1 27.6 40.9 15.0 24.2 33.2 5 36.7 53.4 65.3 19.1 31.8 52.8 15.7 28.2 38.6 13.0 25.6 34.5 6 23.2 30.8 62.7 17.0 35.4 49.0 15.8 29.6 39.6 14.4 25.4 33.4 Mean response ± SEM (mN) 30.4±2.4 47.2±4.3 68.8±3.4 17.9±0.4 34.0±0.9 51.1±0.7 16.3±0.8 28.3±0.4 39.9±0.3 14.0±0.5 24.7±0.6 33.6±0.4 Mean decrease in response ± SEM (mN)

- - - 12.6±2.1 13.2±4.4 17.8±3.2 14.1±2.3 18.9±4.4 28.9±3.4 16.4±2.6 22.5±4.6 35.3±3.5

% age decrease in response - - - 41.4 28.0 25.9 46.4 40.0 42.0 53.9 47.7 51.3 P – value

0.002** 0.030* 0.003** 0.002** 0.008** 0.000**** 0.001*** 0.005** 0.000****



Figure7:Concentration response curve of carbachol-induced contraction on ovalbumin-sensitized guinea pig tracheal tissue in the presence of thymoquinone and propranolol. The data represents the mean ±SEM (n=6). The maximum response with carbachol in sensitized tissue was assumed to be 100%.

Figure 7: Concentration response curve of carbachol-induced contraction on ovalbumin-sensitized guinea pig tracheal tissue in the presence of thymoquinone and propranolol. The data represents the mean ±SEM (n=6). The maximum response with carbachol in sensitized tissue was assumed to be 100%.

Figure 8: Concentration response curve of carbachol-induced contraction on ovalbumin-sensitized guinea pig tracheal tissue in the presence of salbutamol (nM).The data represents mean ±SEM (n=6). The maximum response with carbachol in sensitized tissue was assumed to be 100%.

Bashir et al. 13 Table 9: Effect of salbutamol (nM) and propranolol (nM) on carbachol (nM) induced contraction of ovalbumin sensitized guinea pig tracheal tissue

Carbachol (nM)




Conc. Carbachol in Nanomolar 80 320 1300 80 320 1300 80 320 1300 80 320 1300 Log Concentration 1.9 2.5 3.1 - - - - - - - - - Response of Experiments (Force of contraction in mN) 1

35.8 58.5 84.2 20.9 41.7 65.8 17.9 33.8 59.4 15.1 30.2 50.3

2 24.0 40.4 67.4 19.9 42.5 66.2 15.2 34.2 60.5 13.6 29.5 48.3 3 29.8 44.4 62.0 21.4 40.8 64.2 17.4 36.7 58.8 15.0 28.5 47.6 4 33.1 55.6 71.4 22.4 39.0 63.5 16.0 33.0 57.3 15.0 30.0 49.0 5 36.7 53.4 65.3 21.0 42.1 65.0 18.4 32.5 55.3 13.9 28.7 48.7 6 23.2 30.8 62.7 20.5 38.4 62.3 18.6 34.5 61.8 14.2 30.0 50.4 Mean response ± SEM (mN) 30.4±2.4 47.2±4.3 68.8±3.4 21.0±0.3 40.8±0.7 64.5±0.6 17.2±0.6 34.1±0.6 58.8±0.9 14.5±0.3 29.5±0.3 49.0±0.5 Mean decrease in response ± SEM (mN)

- - - 9.4±2.2 6.4±4.1 4.3±3.1 13.2±2.3 13.1±4.6 10.0±3.5 16.0±2.3 17.7±4.3 19.8±3.2

% age decrease in response - - - 30.9 13.6 6.3 43.4 27.8 14.5 52.6 37.5 28.8 P - value - - - 0.008 0.180 0.225 0.002* 0.037* 0.037* 0.001*** 0.009** 0.002**

Results of six experiments with mean ± SEM, mean decrease in response, % decrease in response as well as level of significance as calculated by two tailed paired sample t-test

Table 10: Relaxant Effect of Thymoquinone and Salbutamol on Ovalbumin Sensitized Guinea Pig Tracheal Tissue

Thymoquinone Thymoquinone +Ovalbumin

C1T1 Vs C1T1 26.6 23.4 C2T1 Vs C2T1 0.5 13.8 C3T1 Vs C3T1 6.2 10.5 C1T2 Vs C1T2 49.2 30.3 C2T2 Vs C2T2 34.1 20.6 C3T2 Vs C3T2 24.3 26.9 C1T3 Vs C1T3 64.1 32.6 C2T3 Vs C2T3 48.1 28.2 C3T3 Vs C3T3 49.4 31.3 C1= 80 nM carbachol T1= 122 µM thymoquinone C2= 320 nM carbachol T2= 244 µM thymoquinone C3= 1300 nM carbachol T3= 488 µM thymoquinone

concentration effect (46.4, 40.0 and 42.0%, respectively). Similarly, the third concentration of 68 nM salbutamol and thymoquinone (concentration of 488 µM) and carbachol (80, 320 and 1300 nM)

reduced the contraction concentrations by 32.6, 28.2 and 31.3%, respectively. While the same concentration of salbutamol thymoquinone and carbachol applied to ovalbumin-sensitized tissue

could not prevent the decreasing concentration effect (53.9, 47.7 and 51.3%, respectively) shown in Figure 11 and supplementary Table 11.


Figure 9: Concentration response curve of carbachol-induced contraction on ovalbumin-sensitized guinea pig tracheal tissue in the presence of salbutamol (nM) and propranolol (nM). The data represent the mean ±SEM (n=6). The maximum response with carbachol in sensitized tissue was assumed to be 100%.

Figure 10: Comparison of the relaxant effect of thymoquinone of the carbachol-induced contraction on normal vs. ovalbumin sensitized guinea pig tracheal tissue. The data represents percentage decrease in response ( n=6).

DISCUSSION The effects of thymoquinone preparations on the inhibition of reactive respiratory disease (inflammation model) were

investigated in the tracheal tissue of guinea pigs. We have shown here that thymoquinone appears to be the most effective extract in connection with anti -inflammatory / immunomodulatory activity. In addition, our results

Bashir et al. 15

Figure 11: Comparison of thymoquinone with salbutamol relaxation of carbachol-induced contraction on normal guinea pig tracheal tissue. The data represents meanpercentage decrease in response (n=6).

Table 11: Comparison of thymoquinone vs salbutamol relaxation of carbachol induced contraction of normal guinea pig tracheal tissue.

Thymoquinone Salbutamol

C1T1 Vs C1S1 26.6 45.3 C2T1 Vs C2S1 0.5 31.8 C3T1 Vs C3S1 6.2 36.1 C1T2 Vs C1S2 49.2 67.2 C2T2 Vs C2S2 34.1 49.6 C3T2 Vs C3S2 24.3 47 C1T3 Vs C1S3 64.1 75.8 C2T3 Vs C2S3 48.1 62.5 C3T3 Vs C3S3 49.4 64.7 C1 = 80 nM carbachol S1 = 17 nM Salbutamol T1 = 122 µM thymoquinone C2 = 320 nM carbachol S2 = 34 nM Salbutamol T2 = 244 µM thymoquinone C3 = 1300 nM carbachol S3 = 68 nM Salbutamol T3 = 488 µM thymoquinone

Data represents mean % decrease in response (n=6).

showed that thymoquinone inhibits the rel ease of some cytokines with inflammatory properties that are found to be upregulated in patients with asthma. Therefore, thymoquinone can have a corticosteroid -like effect in upregulating some infl ammatory mediators to induce remission of asthmatic symptoms. This study enabled us to

identify the most active thymoquinone supplements in various in vivo models of asthma. Other studies showed similar results, which confirm the results of our study. One study investigated the preventive effect of Nigella sativa extract on pneumonia in sensitized guinea pigs. The results showed a decrease in the pathological changes in the lungs


and in the conc entrations of inflammation mediators such as IL-4 in bronchoalveolar lava fluid (Boskabady et al., 2011). Reportedly, Nigella sativa showed a relaxing effect on guinea pig tracheal chains and this effect was significantly higher than that of theophylline. In addition, pretreatment of animals with Nigella sativa reduced tracheal decreased response to cigarette smoke (Keyhanmanesh et al., 2013; Keyhanmanesh et al., 2014b). The use of Nigella sativa extract in various concentrations improved spirometry parameters in asthmatics who were similar to theophylline. In addition, clinical symptoms, namely wheezing and recurrent asthma attacks, were reduced in patients treated with Nigella sativa compared to the control group (Boskabady et al., 2010a).In our study ovalbumin has been utilized as an allergen/ antigen for sensitization and as a challenge to produce airway inflammatory model. It has certain advantages over other antigens in that it is relatively cheap, purified, and made without endotoxins and proteases. Mostly, ovalbumin is combined with an adjuvant to cause allergic airway inflammation. Aluminum hydroxide is usually added as an adjuvant with ovalbumin for better absorption (Conrad et al., 2009; He et al., 2015; Kanagaratham et al., 2018). We compared the percentage of relaxation produced by thymoquinone and salbutamol, the bronchodilator most commonly used in asthma. In this study, we attempted to investigate the likely mechanism of action of thymoquinone by predisposing the tracheal tissue of our test model to carbachol, a muscarinic agonist that causes contractions in

tissue, and propranolol which blocks receptors(Morales et al., 2014).Carbachol is often used in experimental studies to induce smooth bronchial muscle contractions (Kai et al., 2019).In the first series of experiments, the response to an increasing concentration of carbachol in the tracheal tissues of normal (non-sensitized) guinea pigs was observed in the presence of three concentrations of thymoquinone. It was observed that thymoquinone produced more relaxation in presence of high concentrations of the agonist. Our observation is in line with the work of Keyhanmanesh and co-workers, in which they observed a relaxing effect of methanolic components of Nigella sativa (thymoquinone) as one of the components responsible for this relaxation(Keyhanmanesh et al., 2014a).Our results suggest that thymoquinone has muscarinic blockage features that is consistent with El Aziz's observations, as they reported in their study that thymoquinone showed a significant decrease in tracheal coil response to histamine and acetylcholine(El-Ebiary et al., 2016; El Aziz et al., 2011).

In the tracheal tissues of the same group of animals, decrease in the response with three concentrations of thymoquinone on carbachol-induced contractions was observed in the presence of propranolol. We observed thymoquinone caused relaxation in presence of propranolol, but did not completely reversed it. It has been suggested that, besides other mechanisms involved in

smooth muscle relaxation, thymoquinone also exhibits 2

agonistic activity. Recently, it was reported that Nigella

sativa extract is effective for early and late prevention of pulmonary fibrosis and inflammation in rats (Poursalehi et al., 2018). The stimulating ef fect of macerated extract from Nigella sativa on β2-adrenoceptors has already been reported, which is further confirmed by different published literature (Boskabady et al., 2004a; Koshak et al., 2018).

In the second series of trials with ovalbumin-sensitized guinea pig isolated tracheal tissues, our results showed a significant decrease in the response with thymoquinone to increasing carbachol concentrations. In comparison, the percentage decrease in response to thymoquinone in tissues of non-sensitized animals was much higher than that in those exposed to ovalbumin. The difference in the percent response compared in our study may also be due to a change in sensitivity or the number of receptors after sensitization with ovalbumin. Therefore, the comparatively smaller percentage decrease in the response of sensitized tissue in our study can also be attributed to an unchanged number of muscarinic receptors. In 1983, Mita, and co-workers, working on ovalbumin-sensitized guinea pig lungs, reported that sensitizing guinea pigs with the antigen resulted in a reduced number of beta-adrenergic receptors. In addition, in the same study, they reported no change in the number of muscarinic receptors compared to control animals (Mita et al., 1983b).

In the presence of carbachol and propranolol in the sensitized isolated guinea pig trachea, a significant reversal of the relaxing effects of thymoquinone was observed. Comparing the reversal of the relaxing effects of thymoquinone in normal and sensitized tracheal tissues, the percentage decrease in response was greater in normal tissues than in sensitized tissues. Our results are consistent with the previous published literature (Boskabady et al., 2007; Mita et al., 1983b; Shahzad et al., 2009).Ovalbumin-sensitized guinea pig tracheal tissues, salbutamol showed a significant decrease in the response to carbachol. However, a significant reversal occurred when propanolol was added. Our results showed the conventional competitive activity between salbutamol and propanolol, with increasing concentrations of agonists (salbutamol) causing a reaction. Our results are consistent with a number of studies on drug antagonism (Könnecke et al., 2017; Short et al., 2012).

The effects of salbutamol, a beta-adrenoceptor agonist, are known to be competitively controlled by beta-adrenoceptor blockers, while carbachol (a muscarinic agonist) and salbutamol are physiological antagonists in various double-innervated organs. When the comparison was made between relaxation generated by salbutamol in the presence of carbachol in non-sensitized and sensitized tracheal tissues, a greater percentage decrease in response was observed in non-sensitized versus sensitized tissues. While a similar reaction pattern was again observed in the presence of propanolol, these results also follow the previous observations (Boskabady et al., 2010b; Mita et al., 1983b).A recent study investigated the effects of levosimendan and thymoquinone (TQ) on lung injury after myocardial ischemia/reperfusion (I/R) and found the increases expression of caspase 3, p53, and Bax in lung

tissue and has a protective effect on lung as distant organ(Sezen et al., 2018).When comparing the percentage decrease in response of thymoquinone and salbutamol in sensitized tracheal tissues, the overall effect of salbutmol was greater than that of thymoquinone. In the presence of propanolol, the overall effect of salbutmol was greater than that of thymoquinone when comparing the percentage decrease in the response of thymoquinone and salbutamol in sensitized tracheal tissues.

Our study suggests that thymoquinone has a relaxing effect on the smooth muscles of the guinea pig trachea. Together with other mechanisms, this can be due to 2agonistic activities. It can be used in conditions that cause hypersensitivity of the respiratory tract as additional therapy. However, more research is needed to determine the exact mechanism of action. CONCLUSION The study concluded that thymoquinone reduced the amplitude of carbachol-induced contractions of the isolated smooth tracheal muscles in normal and ovalbumin-sensitized guinea pigs. However, beta-blockers do not completely block the effects of thymoquinone. The likely mechanism of thymoquinone decrease in contraction amplitude is based, among other things, on beta and muscarinic receptors. Further studies are needed to determine the exact mechanism of action. ACKNOWLEDGEMENT The authors thank the Department of Pharmacology at th e University of Heal th Sciences in Lahore, Pakistan, for the support they have provided. CONFLICT OF INTEREST The authors have no conflicts of interest to disclose.

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